Cholera is an acute form of diarrhoeal disease that plagued human
civilization over the centuries. The sudden and explosive onset of the
disease in the form of an outbreak or epidemic, coupled with high
mortality and morbidity rates, had a tragic impact on the personal as
well as social life of people living in the affected areas. The enormity
of human sufferings led clinicians and scientists to carry out extensive
research on cholera and Vibrio cholerae (the causative bacterium of the
disease) leading to major discoveries that opened up novel areas of
research or new disciplines in biomedical sciences. An attempt is made
here to summarize some of these breakthroughs and outline their
significance in broader perspectives. Finally, the possible impact of
the global socio-political scenario on the spread of cholera epidemics
(pandemicity

Name: Indian Journal of Medical Research Publisher: Indian Council of Medical Research Audience: Academic Format: Magazine/Journal Subject: Biological sciences; Health Copyright: COPYRIGHT 2011 Indian Council of Medical
Research ISSN:0971-5916

Issue:

Date: Feb, 2011 Source Volume: 133 Source Issue: 2

Topic:

Event Code: 290 Public affairs

Geographic:

Geographic Scope: India Geographic Code: 9INDI India

Accession Number:

252944927

Full Text:

Introduction

Cholera is an acute type of diarrheal illness that affected
millions of people around the world over the centuries. Historically
speaking, there are very few diseases that can match cholera in terms of
its severity and explosive onset in the form of an outbreak or epidemic.
Further, high mortality and morbidity rates associated with classical
cholera had a tremendous tragic impact on the personal as well as social
life of people living in the affected areas. As a consequence, the
disease had found its place in the contemporary literary works in a
number of instances where subtle intricacies of human relationship were
craftily dealt with in the backdrop of an ongoing epidemic (1-3).

The enormity of human sufferings also led clinicians and scientists
to carry out extensive research to formulate effective treatment of
cholera patients as well as to develop strategies for the prevention of
the outbreak of the disease. As a result, considerable progress was made
toward the goal through generation of novel information on the
physiology, genomics and evolution of Vibrio cholerae (the causative
bacterium of the disease), the mechanism of its survival within the host
as well as in the environment, the transmission cycle of cholera, the
pathophysiological and immunological aspects of host -bacterium
interaction, etc. Some of the discoveries were of such profound
importance that these opened up novel areas of research or did herald
new disciplines in biomedical science, particularly those related to
infectious diseases. An attempt is made here to summarize some of these
breakthroughs that were achieved through studies on cholera or V.
cholerae and outline their significance in broader perspectives.

Vibrio cholerae, cholera, Robert Koch and the development of
Medical Microbiology

In the early period of nineteenth century, cholera was believed to
be caused by "miasma" (bad air). In fact, this was one of the
reasons which led people to overlook the early work of the Italian
scientist Fillipo Pacini who had proposed the germ-theory of cholera and
identified the comma shaped organism as the cause of the disease in
1854. In 1883 cholera broke out as an epidemic in Egypt. Fearing that
the epidemic might move further and take a grip over Europe, the German
government sent a medical team to Egypt which included the German
scientist Robert Koch. By the time Koch and his colleagues started their
investigation in Alexandria (Egypt), the epidemic started subsiding.
This prompted Koch to travel to Calcutta (now Kolkata, India) where the
epidemic was still continuing. Investigations carried out with cholera
patients led him to identify the comma shaped cholera bacillus (Vibrio
cholerae, later on named as Vibrio cholerae Pacini 1854) as the
causative agent of the disease (4). In an announcement made in 1884, he
also claimed the isolation of the organism in pure cultures from the
stool of cholera patients, while it was absent in the stool samples from
cases with diarrhoea unrelated to cholera. These observations were in
conformity with two of the four postulates (known as "Koch's
postulates") formulated by Koch himself in 1882 to establish the
microbial aetiology of infectious diseases (5). In order to fulfill the
criteria laid down in the remaining two of his postulates, Koch tried to
infect animals with pure cultures of the organism with little success.
He rightly concluded that the animals were not susceptible to cholera
and took recourse to the extreme step of infecting himself by drinking
pure cultures. However, he came down with only a mild episode of
diarrhoea, an outcome which was later on exploited by his opponents to
ridicule him. Despite this initial failure, human volunteer studies
carried out during the last few decades by various groups of scientists
clearly demonstrated that it is possible to induce cholera in humans by
oral administration of V. cholerae thereby giving further credence to
Koch's postulates, the basic tenets of which hold good even today.
One may not be far away from the truth while concluding that the
pioneering work of Robert Koch on tuberculosis, anthrax and,
subsequently, on cholera laid the foundation stone of Medical
Microbiology, a discipline primarily dedicated to the understanding of
the cause, mechanism of dissemination as well as control of infectious
diseases.

Cholera, Broad Street pump, John Snow and the science of
Epidemiology

In August 1854, cholera broke out in the neighbourhood of London
and several hundred people died within a span of couple of days. John
Snow, a British physician (anaesthesiologist) who had believed that the
disease affecting the gut could be acquired through ingestion of the
causative material, immediately grabbed the opportunity to investigate
the cases which primarily affected people living in a small area near
the Broad Street. After thorough investigation of cases, their location
and source of drinking water, Snow came to the conclusion that a pump
located in Broad Street had to be the source of cholera (6). He met the
authorities and persuaded them to remove the handle of the pump. Once
the handle was removed, cholera cases started declining and the epidemic
was soon over. Further investigation revealed that the pump was
contaminated with infected material from a nearby sewer which led to the
explosive form of the outbreak. The remarkable success of John
Snow's investigation assumes more significance due to the fact that
during his life time (1813-1858) neither the causative organism V.
cholerae was discovered nor the microbial origin of cholera firmly
established. As a matter of fact, Snow's method of investigation of
an epidemic based on systematic collection of data on the incidence or
number of cases, their temporal as well as geographical distribution
patterns, collection of history on individual basis or group-wise and,
finally, the use of statistical methodologies for the analyses of data
opened up an entirely new approach in medical science that gave birth to
the subject of "Epidemiology".

Oral redydration solution for the treatment of cholera: A simple
solution leading to a major medical discovery in the 20th century

Death in "Asiatic cholera" was attributed to the rapid
onset of severe dehydration as a result of massive loss of salt and
water in the form of diarrhoea (cholera gravis). While administration of
saline by the intravenous route, practiced in the nineteenth and
twentieth centuries, was able to reduce the mortality rate dramatically,
the oral route was found to be ineffective. It was soon evident that
patients were not able to absorb sodium chloride when the solution was
administered orally. However, there was an imperative need to develop an
oral rehydration solution (ORS) based treatment regimen that would help
in the clinical management of cases, particularly in the developing
world during the time of epidemic. This led scientists and clinicians to
vigorously pursue their effort to find a solution to the problem (7).
Finally, a simple yet far reaching observation that glucose added to the
ORS was fully absorbed as well as enhanced the absorption of sodium
revolutionized the concept of oral rehydration therapy (ORT). It is
pertinent to note that the development of the concept and its successful
application was an ideal example of partnership between the basic
(physiologic) and applied (clinical medicine) research culminating in a
discovery that was hailed by the medical journal Lancet as
"potentially the most important medical advance in the 20th
century" (8). It is noteworthy that the use of ORT significantly
reduced the mortality and morbidity associated not only with cholera but
also with certain other dehydrating illness thereby saving the lives of
millions of children around the world.

That cholera is caused by toxic factors released by V. cholerae was
first proposed by no other person than Robert Koch himself. However, for
some reasons, it took several decades before the Indian scientist S.N.
De, who workied at Calcutta Medical College, established the role of
cholera toxin in the causation of diarrhoea in the rabbit ileal loop
model (9). In the same year (1959), another Indian scientist N. K. Dutta
and his colleagues, who were attached to Haffkine Institute at Bombay
(now Mumbai), independently reported similar observations (10). While De
used cell-free culture filtrates to induce diarrhoeagenic response in
his model, Dutta used cell-free lysates to demonstrate diarrhoea in the
infant rabbit model. These discoveries stimulated a lot of interest in
the area as a number of scientists were engaged in the purification and
characterization of the CT molecule for structure and function studies
(11). Thus, CT was found to be composed of one A subunit and five B
subunit polypeptide chains with [AB.sub.5] structure. These studies also
revealed that the toxin acts on the target cell by ADP-ribosylation of a
G-protein (GTP-binding protein) that locks the membrane bound enzyme
adenylate cyclase in an active conformation. Continued activation of the
enzyme leads to an elevation in the level of intracellular cAMP thereby
resulting into the loss of salt and water in the form of massive
diarrhoea. Since its discovery, cholera toxin has served as a useful
model to study the mode of action not only of many bacterial toxins but
also of certain polypeptide hormones, growth factors and related
molecules of considerable biological interest.

Viable but nonculturable vibrios: An enigma to bacteriologists

Over the years, microbiologists used in vitro culture and growth
properties of microorganisms as the "gold standard" to
demonstrate the presence of viable bacteria in a given specimen.
Therefore, the observation made by Xu and coworkers (12) that V.
cholerae exists in the environment in "viable but
nonculturable" (VBNC) form was initially received with a lot of
skepticism by the community. Although there were sporadic reports about
the nonculturability of viable bacteria, the VBNC story assumed
considerable significance due to its public health importance. In fact,
a possible explanation for the failure to isolate V. cholerae O1
organisms from the aquatic environment during the inter-epidemic period
was attributable to the presence of these bacteria in the VBNC state.
While the precise mechanism for the existence of the V. cholerae in the
VBNC state is yet to be understood fully, possible inducible factors in
the environment may include poor availability of nutrient, temperature,
salinity, etc (13). Recent studies (14) have shown that VBNC form of V.
cholerae, which exists in coccoid shape and retains basal level of
metabolic activity, can revert to the culturable and infective form
through animal passages. Therefore, the ability of V. cholerae to
survive in the environment in the viable but nonculturable form in
biofilms, and their association with zooplanktons have provided a new
dimension to the ecology of V. cholerae that has a profound impact on
our understanding of the transmission dynamics of cholera.

V. cholerae genome: "A tale of two chromosomes"

Bacterial cells usually contain one circular chromosome which
undergoes bidirectional replication for duplication and distribution to
daughter cells. Therefore, it was a moment of great surprise when a
group of workers demonstrated that the organism V. cholerae (15)
contained two circular chromosomes in their genome. Whole genome
sequence of V. cholerae revealed that most of the genes required for the
pathogenicity and growth of the organism are located on the large
chromosome (chr I) while the small chromosome (chr II) contains genes
required for transcriptional regulation and transport of substrates that
are important for the assimilation of nutrients from the environment
(16). Distribution of genes in two different chromosomes is believed to
facilitate the survival of V. cholerae in human intestine as well as in
the environment through a complex life cycle as a free living organism
and/or in association with biotic or abiotic surfaces. Genomic analyses
of V. cholerae also led to an intriguing hypothesis of generation of
single chromosomal non-replicating cells ("drone cells") that
might aid to the survival of normal (double chromosome) cells under
nutrient deficient conditions through secretion of biodegrading enzymes.
Thus, V. cholerae serves as a model for certain other multichromosomal
bacteria that exist in life cycles interacting with diverse
microenvironments. Further, experimental data generated so far to
understand the mechanism of chromosome replication and segregation in
bacteria with divided genomes have been primarily obtained using V.
cholerae as a model organism (17).

Co-ordinately regulated expression of virulence genes in V.
cholerae and development of the concept of "regulon" in
bacterial pathogenesis

In order to survive under different environmental conditions, V.
cholerae has evolved an intricate signal transduction mechanism that
couples environmental stimuli to a complex regulatory network of gene
expression or "regulon" through a multi-component regulator
system. The work on V. cholerae provided a major boost to expand the
concept of "operon" (linked genes under the control of a
single promoter) to the "regulon" network concept involving
multiple genes and operons under the control of a few regulatory
proteins. Thus, the co-ordinately regulated expression of a set of
virulence genes, which included genes for cholera toxin and colonization
pilus, was shown to be triggered by the regulatory cascade under the
control of the transmembrane protein ToxR and certain other regulatory
proteins, collectively termed as "ToxR regulon" (18). The
availability of the whole genome sequence of V. cholerae has allowed
researchers to unravel the complexities of the regulon system through
microarray based transcriptome analyses of cells isolated either from
cholera patients (19) or from its natural habitat in aquatic
environment. Interestingly enough, the concept of co-ordinately
regulated virulence gene expression in V. cholerae has been found to be
a paradigm feature in the pathogenesis of several other bacterial
pathogens.

Although V. cholerae strains are of common occurrence in their
natural habitat of aquatic ecosystem, not all of these are pathogenic to
humans. Out of V. cholerae strains belonging to more than 200
serogroups, only the O1 and O139 serogroups are known to be associated
with major outbreaks and epidemics of cholera. In recent years, the
mechanism of the evolution of pathogenic strains of V. cholerae has been
an area of intense research. The recently emerged O139 strain (20) has
been shown to be evolved from an O1 El Tor strain primarily through the
exchange of O-antigen biosynthesis genes involving horizontal gene
transfer mechanism (21). Evidences are accumulating that suggest that
pathogenic strains evolved from the nonpathogenic progenitors through
acquisition of virulence-associated genes or gene clusters that are
mobile in nature (22). These include CTX genetic element encoding the
genes for CT and other accessory toxins, vibrio pathogenicity islands
(VPIs) responsible for the synthesis and expression of the major type of
adhesion pili (toxin coregulated pilus or TCP) and certain other
virulence factors, etc. The CTX element was shown to be of bacteriophage
origin (CTXphi) while TCP acted as its receptor (23). Interestingly,
VPI-1 responsible for the expression of TCP was also claimed to be of
another bacteriophage origin (VPIphi), thus raising the intriguing
possibility of one bacteriophage acting as the receptor of another phage
(24). However, in absence of convincing data for the existence of
VPIphi, one would tend to argue that the VPIs were probably acquired by
V. cholerae through horizontal gene transfer process, the mechanism of
which is yet to be clarified. Nevertheless, it would be pertinent to
assume that bacteriophages and pathogenicity islands play an important
role in the mobilization and/or acquisition of gene segments encoding
virulence traits resulting in the evolution of pathogenic bacteria which
include V. cholerae as well.

There has been a genuine concern that growing climatic changes as a
result of global warming are likely to provide a favourable environment
for the propagation of disease-causing organisms that may trigger the
emergence or re-emergence of infectious diseases around the world. The
first major breakthrough in support of this view came through a
retrospective study using data on the seasonal incidence of cholera
cases in Bangladesh and climatological changes. Using a mathematical
model, collaborating scientists from the USA, UK and Spain predicted
that the start of an El Nino event in the equatorial Pacific can lead to
a surge in the number of cholera cases 11 months later in Bangladesh
located several thousand miles away (25). However, a more complex model
had to be developed for the bimodal distribution of cholera cases in a
year that could be linked to seasonal variations in local climate,
sea-surface temperatures and increased population of planktons
supporting the survival and growth of V. cholerae. Since the
transmission dynamics of cholera bears some similarities with certain
types of vector-borne diseases, it would be of interest to determine the
El Nino effect on the outbreaks of diseases like malaria, dengue, etc
(26). Information generated through the cholera model, thus, provides an
opportunity to study other climate sensitive diseases that might lead to
the development of appropriate methodologies linking climatic changes to
disease outbreak prediction and control (27).

"Live" vaccines are dying (?) while "dead"
vaccines are still alive (!): The story of vaccination against cholera

A major emphasis in the area of cholera research was toward the
development of a vaccine against the disease. Pioneering work in this
regard was done by Louis Pasteur as early as in the year 1880. While
working on chicken cholera, Pasteur made a serendipitous observation
that led to the development of the concept and methodologies of
"attenuation" of virulent organisms and their possible use as
"live" vaccines to prevent infectious diseases like cholera,
anthrax, rabies, etc. Surprisingly, despite Pasteur's approach of
vaccinating chickens with live (attenuated) organism, early vaccines
against human cholera were based on dead (inactivated) organisms
administered by the parenteral route. These vaccines, though provided
partial protection, were of limited efficacies to merit further
continuation. It was soon realized that more effective stimulation of
mucosal immunity in the gut could be achieved through oral rather than
parenteral administration of cholera vaccine. Further, availability of
recombinant DNA technology provided the opportunity to develop live,
attenuated V. cholerae strains as candidate oral vaccines capable of
replicating in the intestine and in the process inducing local immunity.
During the past few decades, different groups of researchers developed
several vaccine strains of V. cholerae, primarily through deletion of
genes encoding one (B) or both A and B subunits of CT. Unfortunately,
majority of these vaccines, when administered orally to human
volunteers, produced mild to moderate diarrhoea and, therefore, were
unsuitable for large scale use (28). Further, concern regarding the
safety of recombinant vaccines was raised following the observation that
the genes encoding CT are carried by a phage that can be acquired and
integrated in the genome of nontoxigenic V. cholerae strains expressing
toxin coregulated pilus or TCP. These and other considerations demanded
further modifications of the vaccine strains to eliminate residual
diarrhoea as well as the possibility of reversal to the virulent
phenotype. Therefore, it is quite understandable that only one live
recombinant vaccine (CVD 103-HgR derived from a V. cholerae O1 strain)
is currently available that reasonably meets the criteria of safety,
immunogenicity and effectiveness in human volunteer trials (29).
However, the vaccine failed to show convincing level of protection
against cholera in a large field trial conducted in Indonesia (30). All
these developments led to the resurgence of the concept of dead
(inactivated) vaccines that may be administrated orally without any
apparent reactogenicity or risk of reversion to virulence. One such
vaccine developed by a Swedish group consists of killed whole cells of
V. cholerae with purified B subunit (recombinant) of CT (31) and the
vaccine exhibited reasonably satisfactory level of protection in field
trials. Interestingly, a cheap and modified version of this inactivated
vaccine without the B subunit, administered orally in a population in
Vietnam, was also shown to be quite effective for a period more than a
year or so (32).

The search for an effective and safe cholera vaccine has been a
long and frustrating one as the disease received considerable attention
of scientists over the span of past several decades. However, the search
has generated several important cues that are likely to be of profound
importance not only for vaccinologists but also for immunologists in
general. These include the demonstration of the importance of mucosal
immunity (more precisely mucosal antibodies) in cholera and related
gut-associated diseases, development of methodologies for optimum
stimulation of the immune response in the gut, the understanding of the
homing pattern of lymphocytes connecting mucosal associated lymphoid
tissues at distant sites and, finally, the relative importance of
antibacterial immunity over antitoxin immunity in cholera and ralated
enterotoxigenic enteropathies. More importantly, cholera vaccine
provides a classic example of a bacterial vaccine that highlights the
merit of the continuing debate on the advantages and disadvantages of a
live (attenuated) versus dead (inactivated) vaccine.

Epilogue-Is cholera a disease of "social inequality"?

Cholera is a preventable disease since it is transmitted through
ingestion of contaminated food and water. Unfortunately, despite
tremendous advancements made in our understanding of the disease and its
causative organism, we have not been able to prevent its spread by
providing clean water and sanitation. As a result, the disease continues
to haunt people living under poor socio-economic conditions. In fact,
the number of countries reporting cholera to the World Health
Organization increased by 20 per cent (to 56) in 2004, and case fatality
rates increased to 2.3 per cent, with rates in some outbreaks reaching
as high as 41 per cent in vulnerable groups (33). The situation
continues to remain grim even today. The reason for this dismal scenario
may, to a large extent, be attributed to the lack or unequal
distribution of health care facilities amongst people living in parts of
Asia, Africa, Latin America, etc. In many countries, considerations
based on race, ethnic origin, caste, religion, colour, etc. led to
social inequalities that divided the state into "haves" and
"have-nots". The point is exemplified by Briggs and Briggs in
their book entitled "Stories in the time of cholera" (34),
where the authors critically analyzed the events leading to the outbreak
of cholera in 1992-93 amongst the backward ethnic groups
("Warao") in the delta region of the Orinoco River in Eastern
Venezuela. The authors described in shocking details the sufferings of
the poor people of indigenous origin who were not exposed to the concept
of personal hygiene or modern treatment facilities (the so called
"unsanitary citizens") as compared to the more affluent
"non-indigenous people" to whom modern health care concepts
and facilities were available. ("sanitary citizens"). The
racial divide probably facilitated the rapid spread of V. cholerae on
its arrival to these islands that prompted the authors to conclude that
"when germs and race mix, however inadvertently, the result is
often fatal". One may add to this combination the role of political
scenario of the affected country as an authoritarian regime often likes
to cover up the failure of its inefficient (or even corrupt)
administrative machinary thereby allowing the continuation of the
epidemic unabated. The recent outbreak of cholera in Zimbabawe in 2008
is a glaring example of utmost callousness shown by such a regime in the
face of an ongoing epidemic that affected a large part of the country
putting half of its population at risk (35). Fortunately, there is often
a silver lining amidst the dark scenario in the form of commendable
efforts put forward by the media, social workers and various aid
agencies to bring the plight of the people to the attention of
international community as well as to initiate relief and other health
care measures to alleviate sufferings of the poor people.

Received June 14, 2010

References

(1.) Marquez GG. Love in the time of cholera. London: Penguin
Books; 1989.